Document Detail


Linkage between fructose 1,6-bisphosphate binding and the dimer-tetramer equilibrium of Escherichia coli glycerol kinase: critical behavior arising from change of ligand stoichiometry.
MedLine Citation:
PMID:  12680779     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Escherichia coli glycerol kinase (EC 2.7.1.30; ATP-glycerol 3-phosphotransferase) is inhibited allosterically by fructose 1,6-bisphosphate (FBP), and this inhibition is a primary mechanism by which glucose controls glycerol utilization in vivo. Earlier work indicates that glycerol kinase displays a dimer-tetramer equilibrium in solution, FBP shifts the equilibrium toward the tetramer, and tetramer formation is required for FBP inhibition. However, equilibrium constants for FBP binding and dimer-tetramer assembly that describe the linkage between these processes are unknown. Here, decreased fluorescence anisotropy of extrinsic fluorophores fluorescein and 2',7'-difluorofluorescein due to homo fluorescence resonance energy transfer (homo-FRET) is used to quantitate tetramer assembly and FBP binding. Glycerol kinase is labeled with extrinsic fluorophores covalently attached to an engineered surface cysteine residue under conditions that prevent labeling of native cysteine residues. Tryptic peptide mapping and MALDI-MS verify labeling at the engineered site only. Initial velocity studies show the labeling does not alter the catalytic properties or FBP inhibition. The steady-state fluorescence anisotropy of enzyme with a labeling stoichiometry of approximately 0.1 mol of fluorophore/mol of subunit is not sensitive to increased protein concentration or binding of FBP, indicating the absence of homo-FRET. However, steady-state fluorescence anisotropy of enzyme with a labeling stoichiometry of approximately 0.4 mol of fluorophore/mol of subunit decreases with increasing protein concentration, which is consistent with depolarization due to homo-FRET. The protein concentration dependence of the decreased fluorescence anisotropy is described by a dimer-tetramer equilibrium with an apparent dissociation constant of 61 +/- 7 nM (subunits) at pH 7.0 and 25 degrees C. FBP binds to both the dimer and tetramer of glycerol kinase, and the FBP concentration dependence of the apparent dissociation constant for the dimer-tetramer equilibrium shows critical behavior. The apparent dissociation constant decreases and then increases with increasing FBP concentration, reaching a minimum at about 20 mM FBP. Critical behavior is seen also in the FBP dependence of the inhibition. The critical behavior arises because tetramer dissociation increases FBP stoichiometry from two sites per tetramer to four half-sites per two dimers. The phenomenological description of the coupling between tetramer assembly and FBP binding shows antagonistic binding of FBP to the two sites on the tetramer, indicating that the strong positive cooperativity observed for FBP inhibition of catalytic activity (Hill coefficient approximately 1.5) is due to the approximately 4000-fold higher affinity of the tetramer for FBP rather than to positive coupling between the two FBP sites.
Authors:
Peng Yu; Donald W Pettigrew
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Biochemistry     Volume:  42     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  2003 Apr 
Date Detail:
Created Date:  2003-04-08     Completed Date:  2003-05-12     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  4243-52     Citation Subset:  IM    
Affiliation:
Department of Biochemistry and Biophysics, Center for Advanced Biomolecular Research, Texas A&M University, College Station, Texas 77843-2128, USA.
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MeSH Terms
Descriptor/Qualifier:
Biopolymers
Escherichia coli / enzymology*
Fructosediphosphates / metabolism*
Glycerol Kinase / chemistry,  metabolism*
Ligands
Models, Molecular
Protein Conformation
Spectrometry, Fluorescence
Grant Support
ID/Acronym/Agency:
GM-49992/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Biopolymers; 0/Fructosediphosphates; 0/Ligands; 488-69-7/fructose-1,6-diphosphate; EC 2.7.1.30/Glycerol Kinase

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